Galaxy Visualisation - Tutorial

Description:

Visualizations may be very helpful in understanding data better. There is a whole range of visualizations, from rather simple scatter and barplots up to projections of high dimensional data or even entire genomes. Many of these visualizations often require a lot of tweaking and changes in settings like zooming in and assigning colors, etc. Therefore, visualizations are ideally interactive, and changing settings is often an initial step in exploring data. For this reason it may be inconvenient to make use of static galaxy tools because it lacks these interactive features. For these situations Galaxy offers the option to create visualizations plugins, file format specific javascripts that integrate with the history menu, without making redundant copies of data.

In this tutorial we shall go through how this system works and create a simple visualization plugin. The tool will create a visualization of the number of aligned reads per chromosome of a BAM file, and we will discuss possible optimizations and advantages and disadvantages of the proposed implementation.

Development in Galaxy

Description:

Galaxy is an open-source project. Everyone can contribute to its development with core Galaxy development, integration of softwares in Galaxy environment, ... Here, you will find some materials to learn how to contribute to Galaxy project.

New perspectives on nitrite-oxidizing bacteria - linking genomes to physiology

Description:

It is a generally accepted characteristic of the biogeochemical nitrogen cycle that nitrification is catalyzed by two distinct clades of microorganisms. First, ammonia-oxidizing bacteria and archaea convert ammonia to nitrite, which subsequently is oxidized to nitrate by nitrite-oxidizing bacteria (NOB). The latter were traditionally perceived as physiologically restricted organisms and were less intensively studied than other nitrogen-cycling microorganisms. This picture is contrasted by new discoveries of an unexpected high diversity of mostly uncultured NOB and a great physiological versatility, which includes complex microbe-microbe interactions and lifestyles outside the nitrogen cycle. Most surprisingly, close relatives to NOB perform complete nitrification (ammonia oxidation to nitrate), a process that had been postulated to occur under conditions selecting for low growth rates but high growth yields.

The existence of Nitrospira species that encode all genes required for ammonia and nitrite oxidation was first detected by metagenomic analyses of an enrichment culture for nitrogen-transforming microorganisms sampled from the anoxic compartment of a recirculating aquaculture system biofilter. Batch incubations and FISH-MAR experiments showed that these Nitrospira indeed formed nitrate from the aerobic oxidation of ammonia, and used the energy derived from complete nitrification for carbon fixation, thus proving that they indeed represented the long-sought-after comammox organisms. Their ammonia monooxygenase (AMO) enzymes were distinct from canonical AMOs, therefore rendering recent horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. Instead, their AMO displayed highest similarities to the “unusual” particulate methane monooxygenase from Crenothrix polyspora, thus shedding new light onto the function of this sequence group. This recognition of a novel AMO type indicates that a whole group of ammonia-oxidizing microorganisms has been overlooked, and will improve our understanding of the environmental abundance and distribution of this functional group. Data mining of publicly available metagenomes already indicated a widespread occurrence in natural and engineered environments like aquifers and paddy soils, and drinking and wastewater treatment systems.

Revealing and analyzing microbial networks: from topology to functional behaviors

Description:

Understanding the interactions between microbial communities and their environment well enough to be able to predict diversity on the basis of physicochemical parameters is a fundamental pursuit of microbial ecology that still eludes us. However, modeling microbial communities is a complicated task, because (i) communities are complex, (ii) most are described qualitatively, and (iii) quantitative understanding of the way communities interacts with their surroundings remains incomplete. Within this seminar, we will illustrate two complementary approaches that aim to overcome these points in different manners.

Holistic metagenomics in marine communities

Description:

Complex microscopic communities are composed of species belonging to all life realms, from single-cell prokaryotes to multicellular eukaryotes of small size. Each component of a community needs to be studied for a full understanding of the functions performed by the whole assemblage, however methods to investigate microbiomes are generally restricted to a single kingdom. Using examples from the Tara Oceans project, we will show how size fractionation and use of varied metabarcoding, metagenomics and metatranscriptomics approaches can help studying the marine plankton community as a whole, in a wide geographic space.

Hidden in the permafrost

Description:

The last decade witnessed the discovery of four families of giant viruses infecting Acanthamoeba. They have genome encoding from 500 to 2000 genes, a large fraction of which encoding proteins of unknown origin. These unique proteins meant to recognize and manipulate the same building blocks as cells raise the question on their origin as well as the role viruses played in the cellular word evolution. The Mimiviridae and the Pandoraviridae are increasingly populated by members from very diverse habitats and are ubiquitous on the planet. After prospecting the space, we went back in the past and isolated two other giant virus families from a 30,000 years old permafrost sample, Pithovirus and Mollivirus sibericum. A metagenomics study of the sample was performed to inventory its biodiversity and assess to what extend the host and the viruses were dominant. I will describe the two sequencing approaches which have been used and compare the results.

EBI metagenomics (EMG, https://www.ebi.ac.uk/metagenomics/) is a freely available hub for the analysis and exploration of metagenomic, metatranscriptomic, amplicon and assembly data. The resource provides rich functional and taxonomic analyses of user-submitted sequences, as well as analysis of publicly available metagenomic datasets held within the European Nucleotide Archive (ENA). EMG has recently undergone rapid expansion, with an over 10-fold increase in data volumes in the first 5 months of 2016. It now houses ~ 50k publicly available data sets, and represents one of the largest collections of analysed metagenomic data. As its data content has grown, EMG has increasingly become a platform for data discovery. To support this process, we have made a series of user-interface improvements, including the classification of projects by biome, presentation of results data for better visualisation and more convenient download, and provision of project level summary files. More recently, we have indexed project metadata for use with the EBI search engine, enabling exploration across different datasets. For example, users are able to search with a particular taxonomic lineage or protein function and discover the projects, samples and sequencing runs in which that lineage or function is found. This functionality allows users to explore associations between biomes, environmental conditions and organisms and functions (e.g., discovering protein coding sequences that correspond to certain enzyme families found in aquatic environments at a given temperature range). Here, we give an overview of the EMG data analysis pipeline and web site, and illustrate the use of the new search facility for data discovery.

MG-RAST has been offering metagenomic analyses since 2007. Over 20,000 researchers have submitted data. I will describe the current MG-RAST implementation and demonstrate some of its capabilities. In the course of the presentation I will highlight several metagenomic pitfalls. MG-RAST: http://metagenomics.anl.gov MG-RAST-APP: http://api.metagenomics.anl.gov/api.html

Fast filtering, mapping and assembly of 16S ribosomal RNA

Description:

The application of next-generation sequencing technologies to RNA or DNA directly extracted from a community of organisms yields a mixture of nucleotide fragments. The task to distinguish amongst these and to further categorize the families of ribosomal RNAs (or any other given marker) is an important step for examining the phylogenetic classification of the constituting species. In this perspective, we have developed a complete bioinformatics suite, called MATAM, capable of handling large sets of reads in a fast and accurate way. MATAM covers all steps of the analysis, from the identification of reads of interest in the raw sequencing data to the reconstruction of the full-length sequences of the marker and alignment to a reference database for taxonomic assignment. Part of MATAM is based on the SortMeRNA software, also developed by the team.

Reconstructing genomes from metagenomes: The holy grail of microbiology

Description:

Shotgun metagenomics provides insights into a larger context of naturally occurring microbial genomes when short reads are assembled into contiguous DNA segments (contigs). Contigs are often orders of magnitude longer than individual sequences, offering improved annotations, and key information about the organization of genes in cognate genomes. Several factors affect the assembly performance, and the feasibility of the assembly-based approaches varies across environments. However, increasing read lengths, novel experimental approaches, advances in computational tools and resources, and improvements in assembly algorithms and pipelines render the assembly-based metagenomic workflow more and more accessible. The utility of metagenomic assembly remarkably increases when contigs are organized into metagenome-assembled genomes (MAGs). Often-novel MAGs frequently provide deeper insights into bacterial lifestyles that would otherwise remain unknown as evidenced by recent discoveries. The increasing rate of the recovery of MAGs presents new opportunities to link environmental distribution patterns of microbial populations and their functional potential, and transforms the field of microbiology by providing a more complete understanding of the microbial life, ecology, and evolution.

Prokaryotic Phylogeny on the Fly: databases and tools for online taxonomic identification

Description:

PPF (Prokaryotic Phylogeny on the Fly) is an automated pipeline allowing to compute molecular phylogenies for prokarotic organisms. It is based on a set of specialized databases devoted to SSU rRNA, the most commonly used marker for bacterial txonomic identification. Those databases are splitted into different subsets using phylogenetic information. The procedure for computing a phylogeny is completely automated. Homologous sequence are first recruited through a BLAST search performed on a sequence (or a set of sequences). Then the homologous sequences detected are aligned using one of the multiple sequence alignment programs provided in the pipeline (MAFFT, MUSCLE or CLUSTALO). The alignment is then filtered using BMGE and a Maximum Likelihood (ML) tree is computed using the program FastTree. The tree can be rooted with an outgroup provided by the user and its leaves are coloured with a scheme related to the taxonomy of the sequences. The main advantage provided by PPF is that its databases are generated using a phylogeny-oriented procedure and and therefore much more efficient for phylogentic analyses that "generic" collections such as SILVA (in the case SSU rRNA) por GenBank. It is therefore much more suited to compute prokaryotic molecular phylogenies than related systems such as the Phylogeny.fr online system. PPF can be accessed online at https://umr5558-bibiserv.univ-lyon1.fr/lebibi/PPF-in.cgi

Dr Jekyll and Mr Hyde: The dual face of metagenomics in phylogenetic analysis

Description:

The aim of this lecture is to present the impact of metagenomics and single-cell genomics on public databases. These new powerful approches allow us to have access to the diversity of life on our planet. However, care has to be taken when using these data for posterior analyses, such as phylogenetic studies, as critical errors can still be present in the databases. This course will incorporate examples taken from real studies, and we will investigate methods used for error detection.

Soil metagenomics, potential and pitfalls

Description:

The soil microorganisms are responsible for a range of critical functions including those that directly affect our quality of life (e.g., antibiotic production and resistance – human and animal health, nitrogen fixation -agriculture, pollutant degradation – environmental bioremediation). Nevertheless, genome structure information has been restricted by a large extent to a small fraction of cultivated species. This limitation can be circumvented now by modern alternative approaches including metagenomics or single cell genomics. Metagenomics includes the data treatment of DNA sequences from many members of the microbial community, in order to either extract a specific microorganism’s genome sequence or to evaluate the community function based on the relative quantities of different gene families. In my talk I will show how these metagenomic datasets can be used to estimate and compare the functional potential of microbial communities from various environments with a special focus on antibiotic resistance genes. However, metagenomic datasets can also in some cases be partially assembled into longer sequences representing microbial genetic structures for trying to correlate different functions to their co-location on the same genetic structure. I will show how the microbial community composition of a natural grassland soil characterized by extremely high microbial diversity could be managed for sequentially attempt to reconstruct some bacterial genomes.

Metagenomics can also be used to exploit the genetic potential of environmental microorganisms. I will present an integrative approach coupling rrs phylochip and high throughput shotgun sequencing to investigate the shift in bacterial community structure and functions after incubation with chitin. In a second step, these functions of potential industrial interest can be discovered by using hybridization of soil metagenomic DNA clones spotted on high density membranes by a mix of oligonucleotide probes designed to target genes encoding for these enzymes. After affiliation of the positive hybridizing spots to the corresponding clones in the metagenomic library the inserts are sequenced, DNA assembled and annotated leading to identify new coding DNA sequences related to genes of interest with a good coverage but a low similarity against closest hits in the databases confirming novelty of the detected and cloned genes.

Multiple Comparative Metagenomics using Multiset k-mer Counting

Description:

Large scale metagenomic projects aim to extract biodiversity knowledge between different environmental conditions. Current methods for comparing microbial communities face important limitations. Those based on taxonomical or functional assignation rely on a small subset of the sequences that can be associated to known organisms. On the other hand, de novo methods, that compare the whole set of sequences, do not scale up on ambitious metagenomic projects.
These limitations motivated the development of a new de novo metagenomic comparative method, called Simka. This method computes a large collection of standard ecology distances by replacing species counts by k-mer counts. Simka scales-up today metagenomic projects thanks to a new parallel k-mer counting strategy on multiple datasets.
Experiments on public Human Microbiome Project datasets demonstrate that Simka captures the essential underlying biological structure. Simka was able to compute in a few hours both qualitative and quantitative ecology distances on hundreds of metagenomic samples (690 samples, 32 billions of reads). We also demonstrate that analyzing metagenomes at the k-mer level is highly correlated with extremely precise de novo comparison techniques which rely on all-versus-all sequences alignment strategy.

Assessing microbial biogeography by using a metagenomic approach

Description:

Soils are highly complex ecosystems and are considered as one of the Earth’s main reservoirs of biological diversity. Bacteria account for a major part of this biodiversity, and it is now clear that such microorganisms have a key role in soil functioning processes. However, environmental factors regulating the diversity of below-ground bacteria still need to be investigated, which limits our understanding of the distribution of such bacteria at various spatial scales. The overall objectives of this study were: (i) to determine the spatial patterning of bacterial community diversity in soils at a broad scale, and (ii) to rank the environmental filters most influencing this distribution.
This study was performed at the scale of the France by using the French Soil Quality Monitoring Network. This network includes more than 2,200 soil samples along a systematic grid sampling. For each soil, bacterial diversity was characterized using a pyrosequencing approach targeting the 16S rRNA genes directly amplified from soil DNA, obtaining more than 18 million of high-quality sequences.
This study provides the first estimates of microbial diversity at the scale of France, with for example, bacterial richness ranging from 555 to 2,007 OTUs (on average: 1,289 OTUs). It also provides the first extensive map of bacterial diversity, as well as of major bacterial taxa, revealing a bacterial heterogeneous and spatially structured distribution at the scale of France. The main factors driving bacterial community distribution are the soil physico-chemical properties (pH, texture...) and land use (forest, grassland, crop system...), evidencing that bacterial spatial distribution at a broad scale depends on local filters such as soil characteristics and land use when regarding the community (quality, composition) as a whole. Moreover, this study also offers a better evaluation of the impact of land uses on soil microbial diversity and taxa, with consequences in terms of sustainability for agricultural systems.

Sequencing 6000 chloroplast genomes : the PhyloAlps project

Description:

Biodiversity is now commonly described by DNA based approches. Several actors are currently using DNA to describe biodiversity, and most of the time they use different genetic markers that is hampering an easy sharing of the accumulated knowledges. Taxonomists rely a lot on the DNA Barcoding initiative, phylogeneticists often prefer markers with better phylogenic properties, and ecologists, with the coming of the DNA metabarcoding, look for a third class of markers easiest to amplify from environmental DNA. Nevertheless they have all the same need of the knowledge accumulated by the others. But having different markers means that the sequecences have been got from different individuals in differente lab, following various protocoles. On that base, building a clean reference database, merging for each species all the available markers becomes a challenge. With the phyloAlps project we implement genome skimming at a large scale and propose it as a new way to set up such universal reference database usable by taxonomists, phylogeneticists, and ecologists. The Phyloalps project is producing for each species of the Alpine flora at least a genome skim composed of six millions of 100bp sequence reads. From such data it is simple to extract all chloroplastic, mitochondrial and nuclear rDNA markers commonely used. Moreover, most of the time we can get access to the complete chloroplast genome sequence and to a shallow sequencing of many nuclear genes. This methodes have already been successfully applied to algeae, insects and others animals. With the new single cell sequencing methods it will be applicable to most of the unicellular organisms. The good question is now : Can we consider the genome skimming as the next-generation DNA barcode ?

Rationale and Tools to look for the unknown in (metagenomic) sequence data

Description:

The interpretation of metagenomic data (environmental, microbiome, etc, ...) usually involves the recognition of sequence similarity with previously identified (micro-organisms). This is for instance the main approach to taxonomical assignments and a starting point to most diversity analyses. When exploring beyond the frontier of known biology, one should expect a large proportion of environmental sequences not exhibiting any significant similarity with known organisms. Notably, this is the case for eukaryotic viruses belonging to new families, for which the proportion of "no match" could reach 90%. Most metagenomics studies tend to ignore this large fraction of sequences that might be the equivalent of "black matter" in Biology. We will present some of the ideas and tools we are using to extract that information from large metagenomics data sets in search of truly unknown microorganisms.

One of the tools, "Seqtinizer", an interactive contig selection/inspection interface will also be presented in the context of "pseudo-metagenomic" projects, where the main organism under genomic study (such as sponges or corals) turns out to be (highly) mixed with an unexpected population of food, passing-by, or symbiotic microorganisms.

Who is doing what on the cheese surface? Overview of the cheese microbial ecosystem functioning by metatranscriptomic analyses

Description:

Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We used an iterative sensory procedure to select a simplified microbial consortium, composed of only nine species (three yeasts and six bacteria), producing the odor of Livarot-type cheese when inoculated in a sterile cheese curd. All the genomes were sequenced in order to determine the functional capacities of the different species and facilitate RNA-Seq data analyses. We followed the ripening process of experimental cheeses made using this consortium during four weeks, by metatranscriptomic and biochemical analyses. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. We next applied the same approach to investigate the activity of the microorganisms in real cheeses, namely Reblochon-style cheeses. This provided useful insights into the physiological changes that occur during cheese ripening, such as changes in energy substrates, anabolic reactions, or stresses.

Exploiting collisions between DNA molecules to characterize the genomic structures of complex communities

Description:

Meta3C is an experimental and computational approach that exploits the physical contacts experienced by DNA molecules sharing the same cellular compartments. These collisions provide a quantitative information that allows interpreting and phasing the genomes present within complex mixes of species without prior knowledge. Not only the exploitation of chromosome physical 3D signatures hold interesting premises regarding solving the genome sequences from discrete species, but it also allows assigning mobile elements such as plasmids or phages to their hosts.

Gut metagenomics in cardiometabolic diseases

Description:

Cardio-metabolic and Nutrition-related diseases (CMDs) represent an enormous burden for health care. They are characterized by very heterogeneous phenotypes progressing with time. It is virtually impossible to predict who will or will not develop cardiovascular comorbidities. There is a clear need to intervene earlier in the natural cycle of the disease, before irreversible tissue damages develop. Predictive tools still remain elusive and environmental factors (food, nutrition, physical activity and psychosocial factors) play major roles in the development of these interrelated pathologies. Poor nutritional environment and lifestyle also promote health deterioration resulting in CMD progression. In the last few years, the characterization of the gut microbiome (i.e. collective bacteria genome) and gut-derived molecules (i.e. metabolites, lipids, inflammatory molecules) has opened up new avenues for the generation of fundamental knowledge regarding putative shared pathways in CMD. The gut microbiome is likely to have an even greater impact than genetic factors given its close relationship with environmental factors. In metabolic disorders, the discoveries that low bacterial gene richness associates with cardiovascular risks stimulate encourage these developments. Due to the complexity of the gut microbiome, and its interactions with human (host) metabolism as well as with the immune system, it is only through integrative analyses where metabolic network models are used as scaffold for analysis that it will be possible to identify markers and shared pathways, which will contribute to improve patient stratification and develop new modes of patient care.

In 2010, the MetaHIT consortium published a 3.3M microbiota gene catalog generated by whole genome shotgun metagenomic sequencing, representing a mixture of bacteria, archaea, parasites and viruses coming from 124 human stool metagenomic samples [Qin et al, Nature 2010].
However most of the genes were fragmented, taxonomically and functionally unknown, making it difficult to define and select biomarkers of interest for genome-wide association studies.
Since that, this human gene catalog was improved multiple times, with the last update by [Li et al, Nature Biotechnology, 2014], which generated a 10M gene catalog using more than 1000 metagenomic samples and including some prevalent human microbe genome available at that time. Along with the catalog update, the scientific community developed new tools to challenge the complexity of this dataset and provided new ways to assemble, annotate, quantify and classify the genes coming from these catalogs.
In this talk we will discuss the main approaches related to the computational treatment of the different gene catalog other the time, illustrated by the different papers that deciphered step by step the hidden information of our microbiota and his link with our health.

Metagenomic studies have gained increasing popularity in the years since the introduction of next generation sequencing. NGS allows for the production of millions of reads for each sample without the intermediate step of cloning. However, just as in the past, the quality of the data generate by this powerful technology depends on sample preparation, library construction and the selection of appropriate sequencing technology and sequencing depth. Here we explore the different variables involved in the process of preparing samples for sequencing analysis including sample collection, DNA extraction and library construction. We also examine the various sequencing technologies deployed for routine metagenomic analysis and considerations for their use in different model systems including humans, mouse and the environment. Future developments such as long-reads will also be discussed to provide a complete picture of important aspects prior to data analyses which play a critical role in the success of metagenomic studies.

Docker Tutorial

Description:

Docker is free software that automates the deployment of applications in software containers executant in isolation. A Docker container, away from traditional virtual machines, requires no separate operating system and not providing any but relies instead on the core functionality and uses the isolation of resources and namespaces separated to isolate the operating system as seen by the application.

HOVERGEN tutorial

Description:

HOVERGEN is a database containing homologous vertebrate protein and nucleotide sequences. It allows to easily select similar gene sequences from a wide range of vertebrates. Hence it becomes particularly useful in comparative genomics, phylogeny and evolutionary studies on a molecular level. HOVERGEN Clean contains only complete sequences which reattach to their family. Hence its library is smaller, but more reliable.

Cross Taxa Tutorial

Description:

How query databases according to complex taxonomic critera

Cross-Taxa allows to retrieve gene families that are shared by a given set of taxa, or which are specific to a set of taxa. It is also possible to select genes families which are associated to a certain set of taxa but which are not found in a second set of taxa. Any taxonomic level can be used.

Searching for sequence: Tutorial

Description:

Quick Search is dedicated to a quick search for sequences or sequence families in the databases available on the PBIL server. It is an alternative to WWW Query which allows more complex queries. Quick Search allows you to retrieve sequences or sequence families associated to a single word without specifying what is this word. You can enter indifferently a keyword, a sequence name or accession number, or a taxa name.

Genomic copy number Tutorial

Description:

We will analyze the copy number variations of a human tumor (parotid gland carcinoma), limited to the chr17, from a WES (whole-exome sequencing) experiment. All genomic coordinates correspond to the 2009 build of the reference human genome (hg19 / GRC37).